The interplay between the DNA damage response and ectonucleotidases modulates tumor response to therapy

Abstract

The extracellular nucleoside adenosine reduces tissue inflammation and is generated by irreversible dephosphorylation of AMP mediated by the ectonucleotidase CD73. The pro-inflammatory nucleotides ATP, NAD⁺ and cGAMP, which are produced in the tumor microenvironment (TME) during therapy-induced immunogenic cell death and activation of innate immune signaling, can be converted into AMP by ectonucleotidases CD39, CD38 and CD203a/ENPP1. Thus, ectonucleotidases shape the TME by converting immune-activating signals into an immune-suppressive one. Ectonucleotidases also hinder the ability of therapies including radiation therapy, which enhance the release of pro-inflammatory nucleotides in the extracellular milieu, to induce immune-mediated tumor rejection. Here we review the immune suppressive effects of adenosine and the role of different ectonucleotidases in modulating anti-tumor immune responses. We discuss emerging opportunities to target adenosine generation and/or its ability to signal via adenosine receptors expressed by immune and cancer cells in the context of combination immunotherapy and radiotherapy.

One sentence summary:

Adenosine generation as a rheostat of DNA damage-induced immune signaling in the tumor microenvironment

Introduction

In the last decade there has been unprecedented progress in leveraging the power of the immune system to treat cancer in patients. For the most part, this progress was the result of the identification of immune checkpoint receptors that inhibit the function of T cells and are co-opted to protect tumors from immune-mediated rejection (1). For the majority of cancer patients, however, blocking these immune checkpoint receptors or their ligands is not sufficient. Multiple strategies aimed at promoting an inflamed tumor mircoenvironment to increase responses to immune checkpoint inhibitors (ICI) have been tested. These include the use of radiotherapy, conventional or targeted chemotherapy, and biological agents that mimic pathogen-derived signals such as Toll-like Receptor (TLR) agonists and oncolytic agents (2). Overall, the success of these combinations has been modest, suggesting that it is essential to identify and target immunosuppressive barriers, some of which can be exacerbated by pro-inflammatory treatments.

A large body of preclinical evidence supports the ability of radiotherapy to increase responses to ICI (3). Mechanistically, radiation induces an immunogenic cell death associated with release of pro-inflammatory signals such as adenosine triphosphate (ATP) (4, 5), and activates canonical pathways of viral defence, mediated by exposure of nuclear and mitochondrial DNA to the cytosolic sensor cyclic GMP-AMP synthase (cGAS). cGAS catalyzes the production of the cyclic dinucleotide cyclic GMP-AMP (cGAMP), which binds to stimulator of IFN genes (STING) leading to the downstream activation of type I interferon (IFN-I) and NF-ΚB pathways in the cancer cells and in innate immune cells in the tumor microenvironment (TME) (6). Activation of IFN-I in dendritic cells (DCs) in the TME of the irradiated tumor has been shown to occur via the uptake of tumor DNA as well as cGAMP, which is exported from irradiated cancer cells to the extracellular compartment (7, 8). Overall, extracellular ATP and cGAS-STING pathways promote cross-presentation of tumor antigens by DCs to T cells with priming and expansion of tumor-specific T cells, a process that is facilitated and sustained by ICI (9).

In the clinic, the ability of radiotherapy to induce/enhance responses to ICI is supported by results of early phase trials (10–13). However, the initial assumption that radiotherapy could be broadly beneficial in enhancing ICI responses has been proven incorrect (14). Although there remain multiple open questions pertaining to the dose and sequencing of radiation with ICI and to the optimal radiation target (15), recent preclinical data highlight the importance of ectonucleotidases expressed in the TME and adenosine signaling as a potential immunosuppressive mechanism that needs to be countered to improve the therapeutic efficacy of combinations of radiotherapy and ICI (16, 17). In this review we provide an overview of ectonucleotidases involved in the degradation of extracellular ATP into adenosine (ADO) and regulation of the cGAS-STING pathway in the TME (Figure 1). In addition, we discuss emerging opportunities to modulate this balance to improve responses to combinations of radiotherapy and other pro-inflammatory agents with ICI.

Figure 1. The immunogenicity of radiotherapy is regulated by ectonucleotidases.

1. Radiotherapy -induced cytosolic DNA leads to activation of cGAS that generates cGAMP, part of which can be exported into the extracellular compartment where it activates STING in immune cells. Downstream IFN-I promotes cross-presenting dendritic cell (DC) activation and priming of anti-tumour T cells. 2. ENPP1 converts cGAMP into AMP, generating a substrate for adenosine generation by CD73. 3. ATP released by irradiated cancer cells binds to P2X7R on DCs, promoting production of pro-inflammatory cytokines. 4. CD39 converts ATP into ADP and AMP which is then converted into adenosine by CD73. Adenosine exerts immune suppression by binding to adenosine receptors A2AR and A2BR on immune cells. 5. Extracellular NAD⁺ also contributes to adenosine generation by sequential conversion into ADPR mediated by CD38, into AMP mediated by ENPP1, and into adenosine via CD73. In addition, NAD⁺ serves as a substrate for ADP-ribosyltransferases (ARTs) including ART1 which mono-ADP-ribosylate (MARylate) the P2X7R on T cells to induce NAD-induced cell death (NICD). Importantly CD38 and CD39 expressed by immune cells can offset NICD and ATP-induced cell death by catabolizing NAD⁺ and ATP respectively, thus providing cytoprotective roles.

Overview of the adenosine pathway

Extracellular purines play a critical role in various processes such as cell death, phagocytosis, inflammasome activation, antigen presentation, and cell migration. Purines activate purinergic receptors, divided into three different families: P1 receptors (P1Rs), P2Y receptors (P2YRs), and P2X receptors (P2XRs). P1Rs are G-protein-coupled receptors (GPCRs) activated by adenosine and comprise four subtypes (A1, A2A, A2B, and A3), while P2YRs are GPCRs activated by di- and triphosphate-nucleotides, including ATP, and include at least eight subtypes. P2XRs are ATP-gated ion channels with seven subtypes. Compared with other P2X receptors, P2XR7 has a relatively low affinity to ATP, and its maximal activity is achieved at ATP concentrations of 100–1000 μM (18).

ADO is a potent immunosuppressive metabolite that carries its function essentially through the activation of A2A and A2B GPCRs expressed on immune cells (Figure 2). In solid tumors, ADO accumulates as a result of the hydrolysis of extracellular ATP by ectonucleotidases, reaching concentrations up to 100 μM (19, 20). Extracellular ATP is generally low in most tissue (10–100 nM) but rises up to 1,000-fold in response to tissue damage, hypoxia, ischemia or cell death (21). This accumulation of extracellular ATP is an important danger signal that helps initiate inflammatory processes mainly via the activation of P2Y and P2X receptors expressed on immune cells (Figure 2). In tumors, extracellular ATP plays a key role in promoting antigen presentation and adaptive anti-tumor responses (reviewed in (22)).

Figure 2. Immune regulation through extracellular adenosine (ADO) and adenosine triphosphate (ATP).

In the tumor microenvironment, ADO activates A2AR and A2BR to promote the production of IL-10 and VEGF by myeloid cells, thereby suppressing antigen presentation. In contrast, ATP activates P2X7R and P2Y2R to enhance production of proinflammatory IL-1β, IL-18, IL-6 and IL-12. In CD8+ effector T cells (CD8), A2AR inhibits IFN-γ, TNF-α and proliferation, while P2X7R and P2X4R promote T cell proliferation and immune memory. A2AR further enhances the suppressive function of T regulatory cells (Treg), while P2X7R inhibits FOXP3 expression. In CD4+ T effector cells (CD4), A2AR inhibits IFN-γ, TNF-α and proliferation, while activation of P2X7R promotes T follicular helper (Tfh) function. ADO inhibits B cells via A2AR, while P2X7R promotes B cell activation. Finally, activation of A2AR and/or P2X7R in natural killer (NK) cells inhibit their function.

Several mechanisms of ATP release have been described. These include pannexin channels (PANX), connexins (CONX), ATP-binding cassette transporters and P2X7 purinergic receptors (23, 24). Extracellular vesicles or microparticles are another characterized pathway for ATP release (25). ATP and adenosine diphosphate (ADP) can also be released by platelets through an exocytosis-mediated mechanism (26). Once in the extracellular environment, ATP can activate purinergic signaling via specialized ion-gated channels named P2X receptors, selective G-protein-coupled receptors named P2Y receptors, or can be hydrolyzed into ADO and its mono- or di-phosphorylated derivatives by specialized ectonucleotidases (27).

CD39 and CD73 constitute key ectonucleotidases which promote the metabolism of pro-inflammatory ATP into immunosuppressive ADO. CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; ENTPD1) hydrolyzes extracellular ATP/ADP into AMP (28), while CD73 converts AMP into extracellular ADO (29). Another important ectonucleotidase able to hydrolyse extracellular ATP is ecto-nucleotide pyrophosphate phosphodiesterase-1 (ENPP1, also known as CD203a, PC-1) (30). Additional ADO-generating pathways involving CD38 and ENNP1 are discussed below.

Pro-inflammatory functions of extracellular ATP

ATP and immunogenic cell death

In the TME, dying cells release ATP through caspase- and PANX1-dependent lysosomal exocytosis to alarm the immune system (31). ATP release is an essential component of immunogenic cell death (ICD), which refers to regulated mechanisms of cell death able to stimulate adaptive immune responses (32). Importantly, various cytotoxic agents as well as radiotherapy can cause ICD and enhance adaptive anti-tumor immunity (reviewed in (33)).

During ICD, extracellular ATP stimulates the recruitment of DCs and macrophages via P2Y2 receptors and cooperates with TLR signaling to activate the NLRP3 inflammasome via P2X7 receptors. Upon ATP-P2X7R and TLR signaling, NLRP3 associates with ASC adaptor protein to recruit and activate caspase-1, ensuing cleavage of pro-IL-1β and pro-IL-18 to their secreted forms. Amongst its pleiotropic effects, IL-1β enhances T cell granzyme B expression and proliferation, while IL-18 stimulates innate lymphocytes and antigen-experienced T cells (34). In support of a critical role for extracellular ATP in anti-tumor immunity, targeted blockade of CD39 significantly enhances the effect of ICI in mice in a mechanism dependent on P2X7R, ASC, NLRP3 and IL-1/IL-18 (35).

ATP and macrophages

Macrophages sense tissue damage through purinergic receptors and migrate toward sources of extracellular ATP via P2Y2 receptors (36). Following stimulation with ATP, macrophages further upregulate phosphatidylserine and release several cytokines, including IL-6, IL-12 and CXCL5 (Figure 2) (37–39). ATP signaling via P2X7R eventually upregulates CD39 expression on macrophages, thereby downregulating pro-inflammatory effects (38). Blocking CD39 on macrophages thus constitutes an effective means to enhance IL-12 and tumour-necrosis factor-α (TNF-α) release, while decreasing IL-10 production (40). P2X7R signaling may also promote gasdermin-D (GSDMD)-dependent pyroptosis of macrophages (41), which has been suggested to promote anti-tumor immunity (42).

ATP and T cells

ATP has different effects on T cells. Upon T cell receptor (TCR) signaling, ATP is rapidly released from activated T cells through PANX1 channels. This release of ATP serves T cells in an autocrine and paracrine manner to amplify intracellular Ca²⁺ signaling by activating P2X4 and P2X7 receptors. Extracellular ATP thus stimulates human T cell proliferation. Notably, blocking P2X4R significantly prevents T cell proliferation and migration (43). In vitro work further suggests that basal ATP release from T cells and P2X4R signaling prime unstimulated T cells and subsequently impact their activation upon antigen encounter (44).

Extracellular ATP also impacts T cell-mediated immunological memory and antigen presentation by activating P2X7R (45). P2X7R signaling in T cells is required for the establishment, maintenance and functionality of long-lived central and tissue-resident memory T cells. Using adoptive transfer studies in LCMV-infected mice of wild type versus P2rx7^−/− TCR-transgenic CD8+ T cells (P14 cells), Borges da Silva et al. observed that P2X7R deficiency was associated with a significant loss of CD62L+ central memory T cells (T_CM) (46). In the early phase of memory T cell differentiation, P2rx7^−/− P14 cells showed a selective decline in memory precursor effector cells (MPECs). Mechanistically, loss of P2X7R signaling in MPECs was associated with significantly reduced mitochondrial respiration and aerobic glycolysis, translating to lower T cell survival and reduced recall expansion in vivo.

P2X7R signaling also regulates the generation and maintenance of tissue-resident memory T cells (T_RM), which represent specialized memory T cells that do not recirculate. In a mouse model of LCMV infection, P2X7R was found to be required to support T_RM generation of adoptively transferred antigen-specific T cells. Mechanistically, P2X7R was shown to enhance expression of TGF-β receptor and CD103 on T_RM cells (47). On the other hand, P2X7R expression may limit the accumulation and function of tumor-infiltrating T cells (TILs). Romagnani et al. notably demonstrated that adoptively transfered tumor-specific T cells with deletion of P2rx7 are more effective at reducing tumor growth in mice (48). Mechanistically, activation of P2X7R induced senescence of the TILs.

Extracellular ATP not only promotes antigen presentation and T cell function, it also suppresses T regulatory cells (T_regs), which express higher levels of P2X7R than conventional T cells. P2X7R signaling in T_regs has been shown to trigger a decrease of FOXP3 protein levels and to render T_regs more susceptible to Th17 conversion (49).

P2X7R signaling also plays an important role in T follicular helper (Tfh) cell responses. During immunization, P2X7R signaling promotes expansion of Tfh cells, antigen-specific CD4+ T cells and germinal centers. In the context of immunopathogenic responses, however, where extracellular ATP levels are presumably higher, P2X7R signaling in Tfh has been proposed to limit Tfh survival via GSDMD-mediated pyroptosis (50). In support of a role for ATP signaling in promoting Tfh, ectopic expression of BCL6, the central regulator of Tfh differentiation, represses CD39 (ENTPD1) transcription. Consistent with these observations, CD39 overexpression in CD4+ T cells decreased Tfh development in vitro. Together, these studies suggest that targeting the CD39-CD73 axis may provide a means to enhance antibody responses, for example following vaccination (51).

ATP and NK cells

Intriguingly, evidence suggest that P2X7R activation on NK cells restricts their anti-tumor activity. In vitro, activation of P2X7R with ATP decreased human NK cell cytotoxicity against K562 target cells (52). Injection of B16 melanoma cells in CD8-depleted WT and P2×7r−/− mice also revealed a role for P2X7R in suppressing NK cell-mediated immune surveillance (53).

ATP and B cells

Activation of the P2X7 receptor by extracellular ATP can modulate B cell receptor signaling and influence B cell activation, proliferation, and differentiation. P2X7R stimulation has been shown to promote B cell activation (54), and B cell expression of CD39 is increased on activation in the presence of ATP(55). The ability of B cells to hydrolyze ATP also contribute to immunosuppression. Tumor-infiltrating B cells indeed contribute to increase immunosuppressive ADO levels (56). Septic B cells have also been shown to suppress macrophage-mediated bacterial killing via A2A receptors (57).

Immunosuppression via extracellular ADO

While extracellular ATP can stimulate both innate and adaptive immune responses, tumors are proficient at hydrolysing ATP into ADO. Accordingly, ectonucleotidases are often upregulated in the TME as a result of tissue hypoxia (58), inflammatory cytokines (e.g. TNF-α) (59) and tissue repair pathways (e.g. TGF-β and EMT) (60). Activation of oncogenic pathways, for instance mediated by mutations in KRAS, BRAF, EGFR or ALK-rearrangement, can furthermore upregulate the expression of ectonucleotidases by tumor cells (61). Additionally, radiotherapy has been shown to increase the expression by cancer cells of CD73 and other ectonucleotidases (16, 17).

ADO receptor signaling

Extracellular ADO exerts its effects thought four specific GPCRs. A1 (ADORA1), A2A (ADORA2A) and A2B (ADORA2B) ADO receptors are highly conserved across mammalian species, while A3 (ADORA3) is more variable (reviewed in (62)). In humans, A1, A2A and A3 receptors bind ADO with high affinity, while A2BR has a significantly lower affinity (K_d of 15 μM) that restricts its activation to pathological conditions when ADO concentration is high, such as may occur in the irradiated TME (16). A2AR and A2BR are generally G_s-coupled and therefore trigger intracellular cAMP accumulation, activating protein kinase A (PKA) and EPAC pathways. In contrast, A1 and A3 receptors are preferentially coupled to G_i/o proteins, inhibiting intracellular cyclic AMP production. A2BR and A3R can furthermore recruit G_q/11 proteins and trigger the phospholipase C (PLC) pathways, while all four receptors can induce mitogen-activated protein kinase (MAPK) and JNK pathways.

Landmark studies from Sitkovsky and colleagues established the critical importance of A2AR signaling for downregulation of systemic inflammation and associated tissue damage. Using experimental models of liver damage and sepsis, Otha et al. demonstrated that global A2AR deficiency in mice was associated with a substantial increase in systemic TNF-α, IFN-γ and IL-12 inflammatory cytokines upon endotoxin injection (63).

Interestingly, inosine produced from extracellular ADO by adenosine deaminase can also activate A2A receptors. In contrast to ADO, however, inosine induces a biased activation of the receptor towards increased ERK1/2 signalling over PKA signaling (64). This may account for the distinct impact of ADO and inosine on tumor immunity, although this has not been strictly investigated. Intriguingly, inosine production by the gut microbiome has been shown to stimulate anti-tumor T cells via A2A receptors when sufficient co-stimulation in provided (65).

ADO and myeloid cells

Myeloid-specific conditional deletion studies have revealed the importance of A2A and A2B receptors in the regulation of innate immunity (66). ADO signaling promotes the expression of tolerogenic cytokines by myeloid cells and suppresses the antigen-presenting function of macrophages and DCs. In murine tumors, targeting A2AR with a small molecule inhibitor, AZD4635, significantly enhances the expression of genes associated with antigen processing (Cd1d2, H2-Ea-ps, Cd207), macrophage and DC function (Fcer1a, Cr2), and upregulates the expression of antigen-presenting MHC class II and costimulatory CD86 molecules on macrophages and DCs (67). Further highlighting the impact of A2AR signaling on antigen presentation, in vitro co-culture assays revealed that human DCs exposed to the adenosine analog 5′-N-ethylcarboxamide adenosine (NECA) are significantly impaired in their ability to cross-prime tumor-specific human T cells (>90% reduction), an effect that is completely reversed by AZD4635.

Activation of A2BR also favors tolerogenic DCs and macrophages. Early work in mouse monocyte-derived DCs revealed that A2BR signaling inhibits TNFα, IL-12 and CD86 expression while increasing IL-10 production (68). More recently, using conditional deletion of A2B in LysM+ or CD11c+ cells, Cekic and colleagues demonstrated that A2BR activation in tumor-infiltrating myeloid cells suppresses CD86 and MHC class II expression, and was associated with a significant increase in B16 tumor growth. Notably, ex vivo assays revealed that A2BR-deficient DCs were significantly more effective in driving antigen-specific T-cell responses (66).

ADO and T cells

The A2AR is significantly upregulated upon T cell activation, rendering T cells susceptible to ADO-mediated immunosuppression. Activation of A2AR suppresses TCR, CD28 and IL-2 signalling, thus inhibiting T cell proliferation, survival and cytokine production. Granzyme-mediated cytotoxic functions, however, appear less affected by A2AR signaling, as demonstrated in CRISPR/Cas9 knockout studies (69). Targeting A2A receptors with selective antagonists notably triggers compensatory upregulation of other immune checkpoints, including PD-1, TIGIT, LAG-3 and TIM-3, thus providing a rationale for combination therapies (70). Recent studies of chimeric antigen receptor (CAR) T cells suggest that A2AR distinctively regulates CD8+ versus CD4+ T cells, and that TNF-α production remains partially suppressed by NECA in A2AR-deficient CAR T cells, suggesting a possible role for A2B receptor in ADO-induced T cell inhibition (69).

In humans, CD73 is expressed on approximately 40–80% of circulating naïve CD8+ T cells and 20–40% of circulating naïve CD4+ T cells. Upon activation, CD73 is downregulated and/or released from the cell surface (71). However, it is re-expressed on a smaller fraction of central and effector memory T cells (72). CD39 expression, on the other hand, is induced in T cells by TCR signaling and RUNX3 transcriptional activity (73). CD39 is further upregulated in exhausted T cells. CD39^high PD-1^high tumor-infiltrating T cells are notably enriched for tumor antigen specificity (74). Chronic LCMV responses in mice suggest a role for CD39-mediated purinergic signaling in the regulation of T cell exhaustion (75). Of interest, a recent report revealed a unique role for IL-4 in downregulating CD39 expression on activated T and B cells (73).

Recently, it has been shown that upon activation, human CD8+ T cells release CD73-containing extracellular vesicles (EVs) that generate ADO as a regulatory mechanism (71). Since inhibition of phospholipase or metalloproteinases failed to prevent the loss of membrane-bound CD73 on activated T cells, EV release appears to be the mechanism behind CD73 downregulation from the cell surface in response to TCR activation. Although it remains unclear whether CD39 is also present in T cell-derived EVs, prior studies reported the release of CD39- or CD73-containing EVs from B cells, T_regs and mesenchymal stromal cells (MSCs) (76, 77). Taken together, this suggests a previously underappreciated role for EV-dependent ADO production in the control of immune responses.

CD73 expression is enriched in antigen-experienced memory T cells (T_MEM), which constitute a functionally distinct subset that declines with age (72). CD73+ T_MEM notably show increased ability to differentiate into T_RM, as demonstrated using adoptive transfer studies during LCMV infection in mice (72). Interestingly, an increased expression of ribosomal genes was also observed in CD73+ T_MEM, a characteristic associated with effector rather than memory T cells. Although it remains to be investigated whether the increased functionality and T_RM differentiation potential of CD73+ T_MEM is related to ADO signaling, CD73 appears to identify circulating T_MEM prone to differentiate into T_RM cells.

In T_regs, ADO signaling through the A2AR promotes proliferation, production of immunosuppressive TGF-β and IL-10, and expression of immune-checkpoint receptors, overall enhancing their immunoregulatory activity in vitro (78). A2AR signaling in vivo has been involved in the emergence of induced T_regs during autoimmune disease (79), and exposing T_regs to an A2AR agonist has been shown to enhance the prevention of ischemia-reperfusion injury in mice following adoptive cell transfer (80).

T_regs can also participate in ATP hydrolysis, although CD39 and CD73 are not uniformly expressed, and rarely co-expressed, on human T_regs. There is no correlation between the expression levels of FOXP3, HELIOS, CD39 and CD73 in peripheral human T_regs (81, 82). During in vitro polarization of naïve T cells into induced T_regs (iT_regs), Gerner et al. (81) recently demonstrated that mTOR signaling and TGF-β/SOX4 signaling were required for CD39 expression on iT_regs. CD73, on the other hand, is generally absent from human T_regs, but can be induced in response to IL-2. Accordingly, T_regs from melanoma patients receiving high-dose IL-2 co-express CD39 and CD73 (83). Similarly, flow-sorted CD4+ CD25hiCD127lo human T_regs stimulated with anti-CD3/CD28 and expanded in the presence of high-dose IL-2 (500 U/mL) co-express CD39 and CD73 and display superior suppressive function when compared to matched ex vivo T_regs that do not express CD73 (82).

ADO and NK cells

In mice conditionally deficient for A2AR in NK cells, the maturation of NK cells is enhanced and terminally mature NK cells display increased effector function (84). In the absence of A2AR signaling, NK cells show reduced expression of IL18R1 and KIT genes, which may in part explain better tumor control (85). Tumor-infiltrating NK cells also upregulate CD73 and this is associated with acquired expression of IL-10 and TGF-β production via STAT3 signaling (86). Ex vivo, NK cells can acquire CD73 expression upon coculture with tumor cells in a contact-dependent manner, in a mechanism that may involve 41BB activation on NK cells, although alternative pathways may also be involved.

ADO and B cells

ADO signaling inhibits human B cell proliferation in vitro when stimulated by CD40L and IL-4 (87). The anti-proliferative effect of ADO on B cells is partly reversed by A3 receptor blockade, but appears independent of A2AR. ADO also inhibits human B cells activation with anti-μ-F(ab’)2, evidenced by reduced phosphorylation of Bruton’s tyrosine kinase (BTK) (56). Interestingly, BTK signaling upregulates CD39 expression on B cells.

Targeting CD73 with the blocking mAb ciforadenant has been reported to increase B cell activation, evidenced by upregulation of CD69, CD83, CD86 and MHC II, and to induce differentiation into plasmablasts (88). Intriguingly, these effects were found to be independent of CD73 enzymatic activity or ADO signaling, suggesting transmission of an activation signal downstream of CD73 when ligated by antibodies, as initially proposed by L. Thompson and colleagues (89).

Alternative pathways of ADO generation

In addition to ATP, other pro-inflammatory nucleotides can be converted into extracellular ADO. This occurs independently of CD39, involving ectonucleotidases CD38 and ENPP1, but still requires CD73 for the final step of catabolizing AMP into ADO.

CD38 and its role in cancer

CD38 was first characterized in T and B lymphocytes, where its expression has been used as a marker of differentiation (90, 91), but it is also expressed by NK and myeloid cells. CD38 has several enzymatic properties, firstly as a cyclase converting NAD⁺ to cyclic adenosine diphosphate ribose (cADPR) and secondly as a glycohydrolase (NADase) converting NAD⁺ and cADPR to ADPR (92).

CD38 is the dominant NADase in mammals making it a major regulator of intracellular and extracellular NAD⁺ levels. Chini and colleagues, who were some of the first to determine the enzymatic properties of CD38, showed that NAD tissue levels are 10–20 fold higher in CD38-deficent compared with CD38-proficient mice (92). CD38-mediated intracellular regulation of NAD⁺ has broad implications for cellular energy transfer, as well as for non-redox reactions including NAD-dependent post translational protein modifications including mono- and poly-ADP-ribosylation, mediated by mono-ADP-ribosyltransferases (ARTs) and poly-ADP-ribose-transferases (PARPs), respectively (93–95). In 2013, Malavasi and coworkers demonstrated that ADPR generated by CD38 is hydrolysed by ENPP1 into AMP, which is further dephosporylated into adenosine by CD73 (96, 97).

CD38 is a driver of tumour progression in many hematopoietic cancers. It is highly expressed by multiple myeloma (MM) cells as well as by several myeloid and lymphocytic leukemia cells. In MM, CD38 antibody treatment has proven to be effective by directly killing tumour cells, as well as by eliminating myeloid-derived suppressor cells (MDSCs), T_regs and regulatory B cells (B_regs), resulting in reduced immune suppression (98). Moreover, in the hypoxic and acidic bone marrow niche, the CD38-mediated non-canonical adenosine pathway is believed to operate more effectively than the classical pathway providing an explanation for the anti-tumour effect of CD38-inhibition in this cancer (99).

The role of CD38 in progression of solid tumors remains incompletely elucidated. Several independent studies have shown an association between CD38 expression and anti-tumour immune responses, although it remains unclear whether CD38 expression promotes or counteracts immune cell activity, which may depend on which cells it is expressed. Analysis of CD38 expression in a TCGA human lung cancer patient cohort showed a positive correlation with cytolytic T cell score (100). Similarly, RNA sequencing and immune fluorescence analysis of biopsies from metastatic castration-resistant prostate cancer (mCRPC) patients revealed that mRNA expression of CD38 was associated with active immune gene signature but also with T cell exhaustion and adenosine signalling. In these samples, CD38 was primarily expressed on MDSCs and B cells whose infiltration was a poor prognostic factor for OS (101). However, an inverse correlation between CD38 expression and prostate cancer progression has also been observed and attributed to metabolic reprogramming of tumour cells due to reduced NAD⁺ pools resulting in reduced proliferation (102). Furthermore, in pre-clinical models of esophageal cancer, CD38+ MDSCs were shown to have higher T cell-suppressive capacity than CD38- MDSCs and promote tumour progression (103). Conversely, in studies of hepatocellular carcinoma (HCC), tumour-infiltrating macrophages were predominantly M1-polarized, secreted pro-inflammatory cytokines and their density correlated with improved prognosis following surgery (104). In line with these findings, in HCC patients treated with ICI the overall response rate was higher in patients with high tumour infiltration of CD38+CD68+ macrophages (105).

CD38 expression on immune and cancer cells is upregulated by soluble factors present in the tumour microenvironment including TNF-α, trans-retinoic acid as well as IFN-γ and IFN-β indicating a physiological response to inflammatory conditions (100, 106). Increased CD38 expression has also been observed in colorectal cancer patients following chemotherapy (107), and in melanoma patients following anti-PD-1 therapy (100). Further, CD38 expression on human and murine breast cancer cells is upregulated by exposure to ionizing radiation in a dose-dependent manner (16).

In preclinical lung cancer models, Chen and colleagues reported that CD38-mediated adenosine production promotes acquired tumour resistance to anti-PD-1/PD-L1 therapy (100). In their models, PD-1/PD-L1-mediated upregulation of CD38 on tumour cells was driven by trans-retinoic acid, and IFN-β and tumour knockdown or therapeutic blockade of CD38 synergized with PD-L1 inhibition to promote CD8 T cell-dependent anti-tumour immunity. They further show that CD38 expression correlated with adenosine production, which was reduced upon anti-CD38 antibody treatment. Translation of such combination of CD38 blocking antibodies (daratumumab and isatuximab) with PD-1/PD-L1 blockade in patients with solid tumours however failed to demonstrate a therapeutic benefit, and some studies were terminated early due to increased mortality in the combination arm compared with ICI alone (108–110). These results suggest the need for an improved understanding of the role of CD38 in the TME.

Through its cyclase function, CD38 generates cADPR, which serves as a second messenger for Ca²⁺ mobilization during T cell activation. Together with the finding that CD38 clusters at the immune synapse upon TCR engagement with antigen-presenting cells, this suggests that CD38 is a regulator of T cell function (111). CD38 has also been shown to promote DC trafficking to lymph nodes in response to chemokines, and in CD38-deficient mice T cell priming and T cell-dependent humoral responses are reduced (112). Thus, CD38 inhibition in the context of ICI could potentially interfere with the priming and/or effector phase of the anti-tumor immune response.

Furthermore, ARTs use extracellular NAD⁺ (eNAD) as a substrate to mono-ADP-ribosylate P2X7R expressed on T cells resulting in NAD-induced cell death (NICD). In preclinical studies this process has been shown to operate in tissues during inflammation to selectively eliminate T_RM that are not antigen-activated, as well as T_regs (113–115). Krebs et al. showed that ART2-mediated mono-ADP-ribosylation of T cells following eNAD exposure was higher in CD38-deficient mice compared to wild type mice (116). While ART2 is not expressed in humans due to premature stop codons, ART1 expression by lung cancer cells has recently been characterized as an immune resistance mechanism in NSCLC (117, 118). In an NSCLC mouse model high expression of ART1 by the cancer cells was associated with reduced CD8 T cell infiltration and mice survival. In vitro, ART1+ cancer cells induced NICD of P2X7R+ CD8 T cells, which was exacerbated by CD38 blockade, indicating a protective role of CD38 expressed by P2X7R+ CD8 T cells against NICD. Consistently, P2X7R+ CD8 T cells infiltrating ART1-expressing human lung tumors were enriched for CD38 expression (118). Thus, in solid tumours, particularly following radiotherapy, where eNAD concentrations are high, the function of CD38 as an NADase confers a cytoprotective role for T cells to avoid NICD. These data suggest the possibility that in patients with ART1-expressing tumours CD38-inhibition in combination with ICI could have a detrimental effect by augmenting NICD of tumour-infiltrating CD8 T cells.

ENPP1 and its role in cancer

ENPP1 generates AMP through hydrolysis of ATP, ADPR or through direct hydrolysis of NAD⁺ into AMP which is subsequently converted into adenosine by CD73 (97). Recently, ENPP1 has been shown to suppress innate immune activation in the TME by hydrolysing extracellular cGAMP (119). cGAMP is synthesized by cGAS upon binding to DNA in the cytosolic compartment, where it is present during viral infection or due to genetic instability and the activation of the DNA damage response in neoplastic cells (120). cGAMP binds with high affinity to the endoplasmatic reticulum receptor STING, which subsequently triggers a signalling pathway resulting in transcription of IFN-I and other pro-inflammatory cytokines. Intracellular cGAMP levels are regulated by continuous cellular import and export mediated by transporters (121, 122), including the newly described ATP binding cassette subfamily C member 1 (ABCC1) transporter (123). Intercellular exchange of cGAMP via gap junctions has also been described (124). To date, the only identified hydrolase of cGAMP is ENPP1 which is an extracellular membrane-bound enzyme. By reducing the ability of chromosomally unstable and DNA-damaged cells to propagate immunogenic signalling via cGAMP, while, through this process producing immunosuppressive adenosine-precursors in the form of AMP, ENPP1 is being increasingly recognized as an immune checkpoint and a potential immunotherapeutic target.

High ENPP1 expression in breast cancer has been associated with poor prognosis (125) and increased bone metastasis (126). Carozza and colleagues recently showed that extracellular cGAMP export from irradiated breast tumours dictates radiation-induced immunogenicity in a STING-dependent manner, in large part through activation of conventional type I DC (cDC1). In the aggressive 4T1 triple negative murine mammary cancer model, genetic deletion of ENPP1 in implanted 4T1 tumours, or administration of a cell impermeable ENPP1 inhibitor, resulted in reduced tumor progression (8). Interestingly, radiation increases surface expression of ENPP1 on 4T1 cells, suggesting that activation of cGAS by radiation is coupled with the induction of a counter-regulatory mechanism aimed at reducing immune activation (16).

Loss of tumour-intrinsic ENPP1 by genetic knockout in 4T1 and other tumors was shown to reduce adenosine production in vitro while in vivo it was associated with increased tumor infiltration of activated T cells and increased response to ICI (119). The anti-tumour or pro-tumour effect of ENNP1 deletion or overexpression, respectively, were reduced in STING-deficient mice indicating that ENNP1 promotes tumour progression primarily by preventing cGAMP activation of STING in host cells. Further, significantly higher ENNP1 cancer cell expression was measured in distant metastasis compared with primary tumours. Interestingly, the opposite expression pattern was observed with CD39 potentially indicating that adenosine generated from cGAMP rather than ATP is dominant in at least some advanced and metastatic cancers.

ADO signaling and DNA damage responses

DNA damage induced by certain cytotoxic drugs, ultraviolet (UV) light and radiation has been shown to trigger the release of extracellular ATP (4, 5), which can then activate P2X7 receptors and promote inflammation and tumor antigen presentation (4, 16). Damaged mitochondrial DNA can be repaired, but it is most often degraded (127), and autophagy has been shown to restrict the activation of pro-inflammatory pathways by mitochondrial DNA (128). Cytotoxic stress and radiation can also induce the expression of ectonucleotidases capable of converting ATP into immunosuppressive ADO (16, 17, 129).

The upregulation of CD39 and CD73 in response to DNA damage has been linked to the transcriptional activity of cAMP response element-binding protein (CREB) and HIF-1a (130). CREB is phosphorylated by ataxia telangiectasia-mutated (ATM) (131) while HIF-1a activity is induced by DNA-dependent protein kinase (DNA-PK), ATM and ataxia telangiectasia and Rad3-related (ATR) kinase (132–134) (Figure 3).

Figure 3. Crosstalk between DNA damage and adenosine signaling.

DNA damage induces upregulation of CD73 and CD39 ectonucleotidases via CREB and HIF-1. Adenosine produced by CD39 and CD73 signals through A2BR leading to the activation of PKA, PKC and ERK pathways, which have been associated with increased DNA damage responses (DDR). DNA damage also promotes TGF-β signaling, which increases ectonucleotidase expression. EMT enhances DDR and also upregulate CD73, which in turn supports EMT via A2BR signaling.

Hence, DNA damage is often associated with upregulation of ectonucleotidases that favor accumulation of extracellular ADO. In turn, extracellular ADO signaling can prevent DNA damage (135). Activation of p53 can induce A2BR signaling (136). In human and mouse pancreatic tumor cells treated with gemcitabine or radiation, A2BR signaling suppresses accumulation of double strand DNA breaks (137). In human lung cancer and glioblastoma cells, A2BR signaling also improves recovery from radiation-induced DNA damage (138, 139).

Several pathways downstream of A2BR activation are associated with the DDR, which may explain the role of A2BR in regulating DNA damage. For instance, A2BR is the only ADO receptor able to activate both PKA and PKC pathways. On the one hand, PKA cooperates with the DNA damage checkpoint CHK1 to restrain mitotic progression (140). On the other hand, PKC phosphorylates CHK2 and histone H2A after DNA damage and plays a critical role in maintaining DNA integrity (141). PKC signaling is also known to be activated early upon radiation and has been shown to promote radio-resistance (142).

A2BR also cooperates with growth factor signaling pathways (143, 144), which have been implicated in the DDR. Accordingly, DNA damage enhances ligand-induced growth factor receptor signaling (145) and ERK activity upregulates proteins involved in non-homologous end joining (NHEJ) and homologous recombinational repair (HRR) (146) (145). EGFR translocation to the nucleus also occurs following IR and enhances DNA repair through DNA-PKs (147). Recent studies revealed that activation of A2B receptor promotes EGFR translocation to the nucleus in human lung cancer cells treated with radiation and supports DNA repair (139).

Another mechanism that may link ADO signaling to the DRR is epithelial to mesenchymal transition (EMT), a biological program involved in wound healing that contributes to metastasis and radio-resistance (148). Both CD73 and A2BR have been associated with increased EMT (60, 144). In lung cancer cells, A2BR promotes EMT by enhancing ERK signaling (144). Cancer cells that undergo EMT display increase DDR, notably through stabilization of CHK1 by the EMT-inducing transcription factor ZEB1 (149). Ultimately, cancer cells that undergo EMT lose their cell-cell adhesion and acquire migratory and invasive properties, allowing them to enter the bloodstream or lymphatic vessels and metastasize.

Finally, TGF-β is an important paracrine factor that triggers EMT (148, 150) as well as the upregulation of both CD39 and CD73 (151, 152). DNA damage activates TGF-β signaling, which in turn upregulates DDR proteins, including ATM and BRCA1 (153). As such, TGF-β plays a critical role in the repair of double-strand break (154). Whether ADO signaling is involved in this process remains to be investigated.

As indicated above, DNA damage is also associated with activation of the cGAS-STING pathway through the release of cGAMP into the extracellular environment (124, 155). However, tumor cells often overexpress ENPP1, which hydrolases cGAMP and thus inhibits STING signaling (155). ENPP1 hydrolysis of cGAMP, ATP and ADPR also increases extracellular AMP levels, thereby promoting ADO signaling (Figure 4).

Figure 4. Ectonucleotidases inhibit the cGAS-STING pathway.

Upon DNA damage, the cytosolic sensor cGAS produces cyclic GMP-AMP (cGAMP), thereby activating STING and the transcription of type I IFNs and pro-inflammatory cytokines via IRF3 and NF-kB. Cytosolic cGAMP is also released via transporters and gap junctions to activate STING in neighbouring cells. Paracrine activation of STING, notably in antigen presenting cells (APC) and cancer cells, is suppressed by ENPP1 that hydrolyses cGAMP into AMP and GMP. ENPP1 and CD39 further hydrolyses extracellular ATP into AMP. Extracellular AMP is also produced from the conversion of NAD⁺ to ADPR by CD38, followed by ADPR hydrolysis by ENPP1. Extracellular AMP is hydrolyzed by CD73 into adenosine (ADO), activating cAMP-elevating A2AR and A2BR, which suppresses inflammatory responses, including by blocking NF-kB activity.

In addition to ENPP1, the CD73-ADO axis can also suppress cGAS-STING (137, 156). Our recent study revealed that cancer cell-intrinsic CD73 protected human and mouse pancreatic tumor cells from DNA damage induced by gemcitabine. While A2B receptor signaling partly reversed this phenotype, CD73 may also promote DDR independently of ADO (157). Remarkably, we observed that CD73-deficient tumor cells released significantly greater cGAMP than CD73-expressing tumor cells and upregulated STING target genes (137). Moreover, deletion of cGAS in tumor cells abrogated the in vivo activity of a highly selective CD73 inhibitor (137). Our findings thus highlight a previously unappreciated role for CD73 in regulating the cGAS-STING pathway. Since ENPP1 hydrolyzes both cGAMP and extracellular ATP, co-targeting ENPP1 and CD73 may be synergistic in decreasing ADO signaling and enhancing STING activation. This may be particularly relevant in the context of radiotherapy, known to upregulate both ecto-nucletotidases.

Shifting the balance in favor of immune activation by inhibiting adenosine generation and/or signaling in the TME to improve ICI therapy

Multiple experimental therapeutics targeting CD73, CD39, ENPP1, or the ADO receptors A2A and A2B are in development and some are undergoing clinical testing (158, 159) (Tables 1–3). Strategically, blocking CD73 activity, the final step for ADO generation, should provide the most efficient way to reduce ADO in the TME, whereas targeting CD39 or ENPP1 should enable the accumulation of immune-activating ATP and cGAMP, respectively. For tumors with high levels of adenosine signaling, such as renal cell carcinoma (RCC) and colorectal cancer (160), blocking A2AR may be the most effective intervention. Overall, the choice will have to be guided by the features of the TME and the type of combination therapy that is used. For example, in the context of radiotherapy, blocking CD39 will increase immune activation by ATP, but this effect could be countered by the generation of ADO from NAD⁺ and cGAMP, which is not dependent on CD39. CD38 plays a role in the alternative pathway of ADO generation, but trials targeting CD38 to intentionally disrupt ADO production have not shown favorable results (108–110), which highlights the need to better understand the complex role of CD38 in immune regulation (118). In addition, given that radiation increases ADO concentration in the TME it may be necessary to block not only A2AR, but also the lower affinity A2B receptor. Below we briefly discuss the results, which have been reported for only a few studies in publications or as meeting abstracts.

Table 1.

Ongoing clinical studies testing ectonucleotidases inhibitors

Trial	Phase	Title	Disease	Intervention	Responsible Party
NCT05431270	Phase 1	Dose Escalation/Expansion Study of PT 199 (anti- CD73 mAb) Administered Alone and in Combination With a PD-1 Inhibitor	Advanced Solid Tumor	PT199 (Anti-CD73 mAb)	Phanes Therapeutics
NCT05270213	Phase 1	RBS2418 Evaluation in Subjects With Unresectable or Metastatic Tumors	Advanced Cancer	RBS2418 (ENPP1 antagonist)	Riboscience, LLC.
NCT04336098	Phase 1	Study of SRF617 in Patients With Advanced Solid Tumors	Advanced Solid Tumor	SRF617 (anti-CD39 mAb) Gemcitabine Nab-Paclitaxel Pembrolizumab	Surface Oncology
NCT04306900	Phase 1	TTX-030 in Combination With Immunotherapy and/or Chemotherapy in Subjects With Advanced Cancers	Advanced Solid Tumor	TTX-030 (anti-CD39 mAb) Budigalimab mFOLFOX6, Docetaxel Nab-Paclitaxel Gemcitabine Pembrolizumab	Trishula Therapeutics, Inc.
NCT03884556	Phase 1	TTX-030 Single Agent and in Combination With Immunotherapy or Chemotherapy for Patients With Advanced Cancers	Solid Tumor Lymphoma	TTX-030 (anti-CD39 mAb) Pembrolizumab Gemcitabine Nab-Paclitaxel	Trishula Therapeutics, Inc.
NCT03773666	Phase 1	A Feasibility Study of Durvalumab +/− Oleclumab as Neoadjuvant Therapy for Muscle-invasive Bladder Cancer (BLASST-2)	Muscle I nvasive Bladder Cancer	Oleclumab (anti-CD73 mAb) Duâlumab	Dana-Farber Cancer Institute
NCT03616886	Phase 1/2	Paclitaxel + Carboplatin + Durvalumab With or Without Oleclumabfor Previously Untreated Locally Recurrent Inoperable or Metastatic TNBC (SYNERGY)	Triple Negative Breast Cancer	Oleclumab (anti-CD73 mAb) Paclitaxel Carboplatin Durvalumab	Jules Bordet Institute

Table 3.

Ongoing clinical studies testing ectonucleotidases and adenosine receptor inhibitors

Trial	Phase	Title	Disease	Intervention	Responsible Party
NCT05177770	Phase 2	Study of SRF617 With AB928 (Etrumadenent) and AB122 (Zimberelimab) in Patients With Metastatic Castration Resistant Prostate Cancer	Metastatic castration- resistant prostate cancer	SRF617 (anti-CD39 mAb) AB928 (A2aR/A2bR antagonist) Zimberelimab	Surface Oncology
NCT04381832	Phase 1/2	Adenosine Receptor Antagonist Combination Therapy for Metastatic Castrate Resistant Prostate Cancer (ARC-6)	Castration Resistant Prostate Cancer	AB928 (A2aR/A2bR antagonist) Zimberelimab Quemliclustat (CD73 antagonist) Enzalutamide, Docetaxel Sacituzumab Govitecan	Arcus Biosciences, Inc.
NCT04089553	Phase 2	An Open-label, Phase II Study of AZD4635 in Patients With Prostate Cancer	Prostate Cancer	AZD4635 (A2aR antagonist) Oleclumab (anti-CD73 mAb) Durvalumab	AstraZeneca
NCT04660812	Phase 1/2	An Open Label Study Evaluating the Efficacy and Safety of Etrumadenant (AB928) Based Treatment Combinations in Participants With Metastatic Colorectal Cancer (ARC-9)	Metastatic Colorectal Cancer	AB928 (A2aR/A2bR antagonist) Zimberelimab FOLFOX-6, Bevacizumab Regorafenib Quemliclustat (CD73 antagonist)	Arcus Biosciences, Inc.
NCT03549000	Phase 1	A Phase I/Ib Study of NZV930 Alone and in Combination With PDR001 and /or NIR178 in Patients With Advanced Malignancies.	Advanced Solid Tumors	NZV930 (anti-CD73 mAb) PDR001 NIR178/PBF-509 (A2aR antagonist)	Novartis Pharmaceuticals
NCT03454451	Phase 1	CPI-006 Alone and in Combination With Ciforadenant and With Pembrolizumab for Patients With Advanced Cancers	Advanced Solid Tumors	CPI-006 (anti-CD73 mAb) Ciforadenant (A2aR antagonist)	Corvus Pharmaceuticals, Inc.

Ectonuclotidases inhibitors

Multiple inhibitors of the ectonucleotidase CD73 are undergoing clinical testing, alone or most often in combination with other therapies, including A2A and/or A2B receptor blockade and ICI (Table 1 and 3). Generally, these agents have shown very manageable toxicity but limited activity as monotherapy in early phase studies (161). However, in a randomized study of stage III NSCLC patients treated with chemoradiation, addition of anti-CD73 antibody oleclumab improved overall response rate (ORR) and progression-free survival (PFS) at 12 months over that achieved with anti-PD-L1 alone (162). These results are consistent with preclinical data and suggest that CD73 blockade may be especially effective in the context of DNA damaging therapy and ICI, as discussed above (16, 137). On the other hand, adding oleclumab to durvalumab plus platinum-doublet chemotherapy failed to show clinical benefit in advanced triple-negative breast cancer in a randomized trial, as reported at ESMO 2022 (163). It is still too early to assess the clinical activity of CD39 and ENPP1-targeting agents but results of phase 1 and 1/2 studies are forthcoming (Table 1).

Adenosine signaling inhibitors

Genetic or pharmacologic targeting of A2AR has been shown to improve responses to ICI in several preclinical studies (19, 164, 165). A caveat of A2AR-KO models is that the physiological functions of A2AR during thymic T cell development are also affected (166). In one study, T cell-selective A2AR-KO was shown to compromise the persistence of tumor-specific memory T cells resulting in the inability of mice to control the progression of melanoma and bladder tumors, an effect mediated by reduced IL7 receptor expression on A2AR-KO T cells (167). However, such negative effects have not been observed with the A2AR antagonists that are currently being evaluated in clinical trials, as monotherapy or more often in combination with anti-PD-1/PD-L1 (158, 161). In fact, results of the RCC expansion cohort of NCT02655822 show that A2AR inhibition restored responses to anti-PD-L1 in patients and that durable clinical benefit was associated with increased intratumoral CD8 T cells (168). Inhibition of A2BR was shown to improve the rejection of mouse tumors resistant to A2AR inhibition (169), supporting its therapeutic targeting in the clinic. The simultaneous inhibition of A2A and A2B receptors has the advantage of preventing A2BR-mediated immunosuppression by myeloid cells (169, 170) and disrupting the pro-tumorigenic, tumor-cell intrinsic functions of A2BR discussed above, including the enhancement of radio-resistance. Based on this rationale we have initiated a clinical trial (NCT05024097) testing the dual A2A/A2B receptor antagonist etrumadenant in combination with short course radiotherapy as neoadjuvant treatment in patients with rectal cancer (Table 2). Etrumadenant will be continued during consolidation chemotherapy and anti-PD-1. Other early phase trials are testing adenosine receptor inhibitors with chemoradiation (NCT04892875) in head and neck cancer or in diseases with high adenosine signaling (160).

Table 2.

Ongoing clinical studies testing adenosine receptor inhibitors

Trial	Phase	Title	Disease	Intervention	Responsible Party
NCT02403193	Phase 1	Trial of PBF-509 and PDR001 in Patients With Advanced Non-small Cell Lung Cancer (NSCLC) (AdenONCO)	NSCLC	PBF-509 (A2aR antagonist) PDR001	Palobiofarma SL
NCT05403385	Phase 2	Study of Inupadenant (EOS100850) With Chemotherapy as Second Line Treatment for Nonsquamous Non-small Cell Lung Cancer	Metastatic NSCLC Stage III NSCLC	• Inupadenant (A2aR antagonist) • Placebo • Carboplatin • Pemetrexed	iTeos Belgium SA
NCT03274479	Phase 1	PBF-1129 in Patients With NSCLC	Locally Advanced or Metastatic NSCLC	PBF-1129 (A2aR antagonist)	Palobiofarma SL
NCT05234307	Phase 1	PBF-1129 and Nivolumab for the Treatment of Recurrent or Metastatic Non-Small Cell Lung Cancer	Metastatic NSCLC Recurrent NSCLC	PBF-1129 (A2aR antagonist) Nivolumab	The Ohio State University
NCT04976660	Phase 1/2	TT-4 as a Single Agent in Subjects With Advanced Selected Solid Tumors	Colorectal Cancer Gastric Cancer Hepatocellular Cancer Pancreatic Cancer	TT-4 (A2aR antagonist)	Tarus Therapeutics, Inc.
NCT05024097	Phase 2	A Phase II Study to Test the Efficacy of AB928 (Dual Adenosine Receptor Antagonist) and AB122 (a PD1 Checkpoint Inhibitor) in Combination With Short Course Radiotherapy and Consolidation Chemotherapy for Rectal Cancer (PANTHER)	Rectal Cancer	AB928 (A2aR/A2bR antagonist) Radiation Therapy FOLFOX AB122	Weill Medical College of Cornell University
NCT02655822	Phase 1/1b	Phase 1/1b Study to Evaluate the Safety and Tolerability of CiforadenantAlone and in Combination With Atezolizumab in Advanced Cancers	Renal Cell Cancer Metastatic Castration Resistant Prostate Cancer	Ciforadenant (A2aR antagonist) Atezolizumab	Corvus Pharmaceuticals, Inc.
NCT05501054	Phase 1 b/2	Phase 1 b/2 Trial of Ipilimumab, Nivolumab, and Ciforadenant (Adenosine A2a Receptor Antagonist) in First- line Advanced Renal Cell Carcinoma.	Renal Cell Carcinoma	• Ipilumumab • Nivolumab • Ciforadenant (A2aR antagonist)	M D Anderson Cancer Center
NCT04969315	Phase 1/2	TT-10 as a Single Agent in Subjects With Advanced Selected Solid Tumors	Renal Cell Cancer Castration Resistant Prostate Cancer NSCLC	TT-10 (A2aR antagonist)	Tarus Therapeutics, Inc.
NCT04895748	Phase 1	DFF332 as a Single Agent and in Combination With Everolimus & Immuno-Oncology Agents in Advanced/Relapsed Renal Cancer & Other Malignancies	Renal Cell Carcinoma	DFF3332, RAD001 PDR001 NIR178/PBF-509 (A2aR antagonist)	Novartis Pharmaceuticals
NCT04892875	Phase 1	A Study of Concurrent Chemoradiation in Combination With or Without PD1 Inhibitor AB122 Adenosine 2a Receptor / Adenosine 2b Receptor Inhibitor AB928 Therapies in Locally Advanced Head and Neck Cancers (PANTHEoN)	Head and Neck Cancer	AB122 AB928 (A2aR/A2bR antagonist) Cisplatin, Radiation Therapy	Vanderbilt-Ingram Cancer Center
NCT05198349	Phase 1	First in Human Study of M1069 in Advanced Solid Tumors	Metastatic or Locally Advanced Unresectable Solid Tumors	M1069 (A2aR/A2bR antagonist)	EMD Serono
NCT04280328	Phase 1	Study of Ciforadenant in Combination With Daratumumab in Patients With Relapsed or Refractory Multiple Myeloma	Multiple Myeloma	Ciforadenant (A2aR antagonist) Daratumumab	Corvus Pharmaceuticals, Inc.
NCT03207867	Phase 2	A Phase 2 Study of NIR178 in Combination With PDR001 in Patients With Solid Tumors and Non-Hodgkin Lymphoma	Advanced Solid Tumors NHL	NIR178/PBF-509 (A2aR antagonist), PDR001	Novartis Pharmaceuticals

Overall, it is likely that a rationale design of clinical trials based on the evaluation of the tumor TME and careful choice of the combinations tested will identify the clinical settings where inhibition of adenosine generation and/or signaling can provide the most benefits.

Conclusions

DNA damage caused by cytotoxic drugs and radiation can elicit acute inflammation in the tumor microenvironment by at least two processes: i) cell death coupled with release of ATP in the extracellular compartment; ii) the accumulation of DNA in the cytoplasm, leading to activation of cGAS with generation of cGAMP, which can be exported from the cancer cells and activate neighbouring antigen-presenting cells, promoting anti-tumor immunity. However, DNA damaging agents can also induce the expression of ectonucleotidases, such as CD39, CD73 and ENPP1, which convert ATP and cGAMP into ADO. Surface receptors for extracellular ADO are expressed by most immune cells, which explains the broadly immunosuppressive effects of ADO. On the other hand, baseline adenosine signaling is variable in different tumors, which may be a reason for the limited single agent activity of various experimental therapeutics targeting CD73, CD39, ENPP1, and ADO receptors A2A and A2B. In addition, pre-clinical and early clinical data suggest that countering ADO-mediated immunesuppression is often insufficient for tumor rejection without additional stimulation of the immune system. However, this class of immuno-oncology drugs may have a significant impact when used in combination with immune checkpoint inhibitors for the treatment of tumors that are spontaneously enriched in adenosine. We therefore propose that blockade of ADO generation and/or ADO signaling has a key role in improving the anti-tumor immune response when used in combination with DNA damaging therapies, which increase extracellular ADO in the tumor microenvironment.

Recent evidence that ectonucleotidases also regulate the ability of tumor cells to repair DNA further stenghten the rationale for targeting CD73 and possibly other ectonucleotidases in combination with DNA damaging therapies. The choice of targeting strategy must be guided by the unique features of each patient tumor and the specific combination therapies employed.

However, information about which biomarkers are informative is still limited, and more work needs to be done. Rational design of clinical trials, based on the evaluation of the TME and careful selection of combination therapies, will be crucial in identifying the optimal clinical settings for inhibition of adenosine generation and/or signaling to maximize therapeutic benefits.